The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabilis and established Mytilopsis sallei

MPB-08432; No of Pages 8 Marine Pollution Bulletin xxx (2017) xxx–xxx

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The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabilis and established Mytilopsis sallei Juan C. Astudillo a,b,⁎, Timothy C. Bonebrake b, Kenneth M.Y. Leung a,b,c,d a

The Swire Institute of Marine Science, Faculty of Science, The University of Hong Kong, Cape d'Aguilar Road, Shek O, Hong Kong, China School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China d Simon F.S. Li Marine Science Laboratory, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China b c

a r t i c l e

i n f o

Article history: Received 30 December 2016 Received in revised form 10 February 2017 Accepted 15 February 2017 Available online xxxx Keywords: Non-native species Temperature Salinity Xenostrobus securis Mytilopsis sallei Brachidontes variabilis

a b s t r a c t The recently introduced bivalve Xenostrobus securis and the previously introduced Mytilopsis sallei (~30 years) are dominant over the native Brachidontes variabilis in estuarine fouling communities in Hong Kong. This study tested whether these introduced species have higher thermal and salinity tolerance than the native species under local subtropical seawater conditions. Survival, attachment, clearance rate and byssal thread production of these three species were examined through 96-h acute temperature and salinity tests. The results indicated that X. securis responded normally over a wide range of temperature and salinity conditions. Though M. sallei exhibited a wide salinity tolerance, its sub-lethal responses decreased in cold-seawater conditions. Brachidontes variabilis had the narrowest tolerance to temperature and salinity. These findings may explain the dominance of the non-native bivalves over B. variabilis. The high tolerance of X. securis enables them to become highly invasive in subtropical regions across Southeast Asia, impacting natural communities and shellfish farming. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Temperature and salinity are two of the most important environmental variables affecting the distribution and abundance of native and non-native marine invertebrates at multiple temporal and spatial scales (Berger and Kharazova, 1997; Canning-Clode et al., 2011; Firth and Williams, 2009). In some cases, non-native species respond better to changes in environmental conditions (e.g. temperature, salinity, low oxygen and pollution) than native species (Dafforn et al., 2009; Groner et al., 2011; Lenz et al., 2011). Based on numerous field observations, marine communities under environmental stress tend to have more non-native species relative to less stressful communities (Astudillo et al., 2014; Dafforn et al., 2009; Occhipinti-Ambrogi and Savini, 2003). Therefore, non-native species with high tolerance to a wider range of abiotic factors, such as temperature and salinity, will likely have a greater ability to settle and establish in novel environments. Hong Kong, located in the subtropical region of the South China Sea, exhibits gradual temperature changes between temperate conditions (~14–18 °C) in winter and tropical conditions (~28–30 °C) in the summer season (EPD, 2013; Morton and Morton, 1983), whereas ⁎ Corresponding author at: The Swire Institute of Marine Science, Faculty of Science, The University of Hong Kong, Cape d'Aguilar Road, Shek O, Hong Kong, China. E-mail address: [email protected] (J.C. Astudillo).

temperature in intertidal pools can fluctuate drastically (~7 °C) during the day (Chan, 2000). The decrease of salinity is intensified in the summer season due to heavy rainfall, especially in estuaries, enclosed bays and artificially enclosed bays (i.e., typhoon shelters) with low seawater exchange (EPD, 2013). This seasonal and spatial variation in environmental conditions can provide a variety of potential habitats for non-native species, but these non-native species may require high environmental tolerance to become invasive and compete with local species that have evolved under these conditions (Melbourne et al., 2007; Shea and Chesson, 2002). Hong Kong harbor is expected to be one of the most invaded places in the world (Seebens et al., 2013). However, due to a lack of studies there are currently few recorded non-native marine species, (Lai et al., 2016). The recorded non-native invertebrates are mainly restricted to fouling communities in sheltered and disturbed areas such as typhoon shelters and estuaries (Astudillo et al., 2014). The Caribbean Mytilopsis sallei (Récluz, 1849) and the Australasian Xenostrobus securis (Lamarck, 1819) are common non-native bivalves occurring in some local fouling communities, where they coexist with the native bivalve Brachidontes variabilis (Krauss, 1848) (Morton and Leung, 2015). Mytilopsis. sallei is invasive in the Indo-Pacific region and was introduced into Hong Kong around 1980, becoming dominant on dockyards within a few years of its introduction (Morton, 1989a). Xenostrobus securis, invasive in the Mediterranean region, Japan and Korea (Kimura and Sekiguchi, 2009; Sousa et al., 2009), has been recently introduced to Hong Kong (first

http://dx.doi.org/10.1016/j.marpolbul.2017.02.046 0025-326X/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046

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J.C. Astudillo et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

seen in 2010) and is becoming more dominant than M. sallei in some fouling communities (Morton and Leung, 2015). Brachidontes variabilis is common on natural and fouling communities in Hong Kong (Morton and Chan, 1990); however, it shows relatively low abundance in the presence of the non-native bivalves (Morton and Leung, 2015), suggesting that it might be at a competitive disadvantage relative to the non-native species. Mytilopsis sallei, X. securis and B. variabilis are brackish water species able to tolerate high salinity fluctuations (Morton and Chan, 1990; Morton and Leung, 2015). These bivalves are found in Hong Kong throughout the year, indicative of their ability to cope with a wide range of temperatures. Acute tests to determine lethal and sub-lethal effects of environmental and pollution conditions are commonly used to assess and compare species' tolerances (Rupp and Parsons, 2004; Verween et al., 2007). Tolerance tests are often used to determine environmental limits of abiotic factors (e.g. temperature and salinity), which can have implications for the spread and success of non-native species (Jofré et al., 2014; Mc Farland et al., 2013; Verween et al., 2007). Bivalves under stress could have behavioral and physiological responses (Berger and Kharazova, 1997; Lockwood and Somero, 2011; Rupp and Parsons, 2004). As bivalves are osmoconformers, their first response to unfavorable conditions is to isolate the tissue by closing their valves, followed by a reduction of functional activity (Berger and Kharazova, 1997; Mc Farland et al., 2013). Sub-lethal responses such as clearance rate and byssal thread production have been considered good indicators of the suitable environmental conditions in which species can reach their optimal physiological responses (Rupp and Parsons, 2004; Sara et al., 2008). The ability of non-native bivalves to attach on the substrata by producing byssal threads and filter food under different environmental conditions must then play an important role in their potential to establish and spread (Babarro and Abad, 2013; Mc Farland et al., 2013). Based on the high abundance and wide distribution of X. securis in estuaries compared to M. sallei and B. variabilis in Hong Kong, it is expected that X. securis has higher environmental tolerance than the other two species. Given this, this study aimed to determine and compare the survival and sub-lethal responses of the non-native and native bivalve species under acute exposure to a range of temperature and salinity combinations present in the marine environment of Hong Kong.

2. Materials and methods 2.1. Bivalve species and collection Mytilopsis sallei and Xenostrobus securis were collected from Kwun Tung pier, Kowloon, Hong Kong inside the typhoon shelter (Fig. 1), whereas Brachidontes variabilis was collected from floating buoys in Tai Tam Bay, Hong Kong Island (Fig. 1) due to its low abundance in Kwun Tong. All bivalves were collected from shallow subtidal fouling communities. The bivalves and fouling communities were scraped from the surfaces, kept in plastic containers with seawater and transported to the laboratory within 4 h. Kwun Tung typhoon shelter is an artificially semi-enclosed system (to protect vessels against rough sea conditions during typhoons) exposed to natural and human disturbances in Hong Kong. It is located in a highly populated and old industrial area, with freshwater input from Kai Tak River and rainwater drainage. Tai Tam Bay is a sheltered bay, surrounded by small villages with freshwater input from streams of Tai Tam Tuk area. Previous environmental data for a similar period collected in both sites indicated that the surface seawater temperature (15.6– 30.2 °C) and salinity (23–33‰) conditions of Tai Tam Bay (Lui 1991) were generally similar to those in Kwun Tong typhoon shelter (16–28.6 °C and 26–33‰) between seasons (data from Environmental Protection Department of the Hong Kong Special Administration Region Government).

2.2. Cleaning and selection The individuals of each bivalve species were carefully detached from the fouling community by cutting their byssal threads with scissors to avoid any damage to their byssal gland. Epibionts were removed from their shells using a small metal scraper and kitchen wipe. Bivalves of 10–20 mm in shell length (anterior-posterior axis) were selected for the experiments because they were the most common size range among the three bivalve species, and this size range also represents reproductive individuals (Abdel-Razek et al., 1993; Ma, 2013; Morton, 1989a).

Fig. 1. Map with the sites where the experimental bivalves were collected: Xenostrobus securis and Mytilopsis sallei in Kwun Tong and Brachidontes variabilis in Tai Tam.

Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046

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2.3. Acclimation The selected bivalves were acclimated to laboratory conditions for 2 weeks before starting the experiments in 20 l plastic aquarium under 12:12 day/night photoperiod, filtered seawater (0.22 μm) with salinity of 30 ± 2‰ and temperature of 22 ± 1 °C (room temperature) and constant air supply via aeration. Every second day the seawater was renewed and the bivalves were fed daily with commercial coral food made of phytoplankton (Plancto, Aqua Medic, Germany). Dead bivalves were removed from the aquarium to avoid degradation of seawater quality. Twenty four hours before starting the experiments the feeding was stopped to avoid high levels of feces and undigested food which would decrease the seawater quality in the testing aquaria (e.g. lowering dissolved oxygen).

of the acute test were transferred to a plastic container with 100 ml of seawater under the same temperature and salinity conditions with 4 replicates per treatment (∑n = 3 temperatures × 6 salinities × 4 replicates = 72 experimental units per species). For each treatment there was a control without bivalves (two replicates) to account for the potential change of neutral red concentration due to factors other than bivalve filtration. After about 20 min from transferring the bivalves (once most of the bivalves started to filter or move their foot), 0.5 ml of a stock solution of neutral red (0.1% in deionized freshwater) was added to each container and stirred gently to reach a testing solution of ~0.0005%. Seawater samples were taken at 0 and 120 min with 0.2 ml micropipette to determine the absorbance. Absorbance was measured with a SpectraMax Plus 384 microplate reader (USA) at 450 nm. The clearance rate was estimated using Quayle's (1948) equation (Coughlan 1969):

2.4. 96-h acute salinity and temperature test The bivalve species were exposed to 96-h acute temperature and salinity conditions to compare their lethal and sub-lethal responses. Individuals of each species were randomly placed in 450 ml plastic aquaria under a combination of three temperatures (14, 22 and 30 °C) and six salinities (7, 12, 17, 22, 27, 32‰) in an orthogonal and balanced design with 4 replicates and 10 bivalve individuals per replicate (∑n = 3 temperatures × 6 salinities × 4 replicates = 72 experimental units per species). Temperature and salinity values represent the seasonal and spatial variation of such seawater parameters in Hong Kong (EPD, 2013). The average and range of temperature and salinity for ten marine zones monitored monthly by the Environmental Protection Department of the Hong Kong Special Administrative Region Government during the recent ten years (2005–2015) are shown in supplementary material (Fig. S1 and Table S1). The temperature treatment of 22 °C and salinity of 32‰ could be considered as a control condition due to the fact that the acclimation was conducted under similar conditions. The temperature treatments were achieved with water baths; 14 °C using a water chiller (HC-300A, Hailea, China), 22 °C at room temperature (air conditioned), and 30 °C with a submersed heater (HT-150, Tetratec, Poland). Experimental seawater salinities were prepared with filtered natural seawater (0.22 μm) and diluted with deionized freshwater. Before starting the experiments, the seawater was oxygenated with an air pump to ensure a saturation of dissolved oxygen. The low and high experimental temperatures (14 and 30 °C) were gradually reached from 22 °C (i.e., control condition) in 2-h in the water bath after the bivalves were added into the aquaria, and then the experimental period was commenced. The experimental aquaria were covered with a transparent plastic film (with small holes for gas exchange) to keep temperature and salinity conditions stable during the experiment. The test was run under 12:12 day/night photoperiod. Each aquarium was checked every 24-h and dead individuals were removed. Bivalves were considered dead when their valves were open, they were not filtering and failed to respond to the touch of a glass rod. Seawater was renewed once after 48-h of the experimentation. 2.5. Survival and number of individuals attached At the end of the 96-h acute test, survivors and individuals attached to the plastic container or to another individual were counted. Individuals that died in each replicate were considered non-attached. After the acute test, the clearance rate and byssal thread production of the survivors were measured using the same temperature and salinity conditions as in the acute test. 2.6. Clearance rate The clearance rate of the bivalves was estimated by measuring removal of suspended neutral red dye (SIGMA, 65% dye content) (Rajagopal et al., 2005). Three random individuals from each treatment

3

m¼

M gt

    C t0 C tc0 loge − loge C t1 C tc1:

where m is filtration rate, M is volume of test solution (ml), g is the total dry weight (g) of the bivalves, Ct0 and Ct1 is the neutral red concentration at the beginning (t0) and at the end (t1) of the testing time (t) respectively, and Ctc0 and Ctc1 is the neutral red concentration in the control at t0 and t1 respectively. The concentration of the neutral red dye was determined with a standard calibration curve built with 0.0001, 0.00025, 0.0005, 0.00075 and 0.001% of neutral red dye. 2.7. Byssal thread production Surviving bivalves of each treatment were randomly chosen to determine byssal thread production. One bivalve was placed in a transparent plastic container with 100 ml (5 replicates) under the same temperature and salinity treatment conditions as the acute test (∑n = 3 temperatures × 6 salinities × 5 replicates = 90 experimental units per species). The containers were covered with a plastic film with small holes to reduce evaporation. After 48-h, the individuals were carefully removed from the container to avoid pulling the byssal threads. Seawater was removed and the container rinsed with a 1% solution of neutral red to dye the byssal threads. Containers were dried in an oven at 60 °C. The number of byssal threads was counted as the number of plaques attached to the wall of the container using a stereoscopic microscope. 2.8. Statistical analysis Two-way analysis of variance (ANOVA) tests were performed to test the effect of temperature (fixed, 3 levels) and salinity (fixed, 6 levels) on survival (number of survivors), attachment (individuals attached), clearance rate and byssal thread production. Analyses were performed separately for each species and variable. Normality was tested with Shapiro-Wilk test and homogeneity of variances with Levene's test. Data were transformed to fulfill the assumption of equal variance when necessary. Data that did not pass homoscedasticity after different transformations were tested using ANOVA with raw data using alpha of 0.01 to reduce type I error (Underwood, 1997). Tukey's HSD post-hoc test was used after the ANOVA. Permutational MANOVA in the software PERMANOVA + for PRIMER v6.0 (Anderson et al., 2008) was used to compare all the three bivalve species (fixed, 3 levels), temperature (fixed, 3 levels) and salinity (fixed, 6 levels) in one multivariate model (i.e., lethal and sub-lethal responses). A posteriori pair-wise comparison was conducted only on the species factor to determine differences between the three bivalve species. Principal coordinates analysis (PCO) was used to summarize the four variables in one multivariate ordination. For the multivariate analysis, each variable was transformed to percentage according to the highest value among the treatments for each species. The PCO analysis was conducted with the average of the

Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046

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replicates of each variable per species. Data were not further transformed because the Draftsman plot indicated that transformations did not improve the scatter and correlation structure of the data. 3. Results Acute temperature and salinity conditions had little effect on the survival of Mytilopsis sallei and Xenostrobus securis after 96-h of exposure (Table 1, Fig. 2). Both non-native species showed survival above 90% across all treatments. The survival of M. sallei showed no significant differences among the treatments (Table 1A, Fig. 2A). Xenostrobus securis indicated a significant difference within temperature and salinity treatments with a slightly decline in their survival at 30 °C and low salinities of 7, 12 and 17‰ (Table 1A, Fig. 2B). The survival of the native Brachidontes variabilis was significantly affected by the interaction of temperature and salinity (Table 1A, Fig. 2C). Their survival at the high temperature treatment (30 °C) decreased with salinities lower than 17‰, dropping to about 30% under salinities of 7 and 12‰. On the other hand, B. variabilis showed high survival (above 95%) in all the salinity treatments at temperatures of 14 and 22 °C. At 14 °C, the number of attached individuals of M. sallei (b10%) was significantly lower than that in the other two temperatures (N 90%), while salinity had no effect on the attachment (Table 1B, Fig. 2A). In X. securis, the interaction of low temperature and salinities below 12‰ significantly reduced their attachment (Table 1B). At 7‰, their attachment dropped below 40% at 22 and 30 °C, whereas at 14 °C their attachment was completely inhibited (Fig. 2B). Xenostrobus securis showed high attachment (N90%) in salinities at or above 17‰. The attachment of B. variabilis was significantly affected by the interaction of temperature and salinity (Table 1B, Fig. 2C). Low temperature reduced their attachment for salinities below 22‰ (from 38 to 0%). Salinities below 17‰ drastically reduced their attachment, and under 12‰ their attachment was greatly inhibited (b 5%). Brachidontes variabilis showed high attachment (N 85%) in salinities above 27‰ across all temperatures. By observation, M. sallei had the lowest clearance rate among the three species (Table 1C, Fig. 3). The clearance rate of M. sallei was significantly reduced at 14 °C (Table 1C, Fig. 3A), being at least 2–3 times lower than the other two temperatures. The clearance rate of X. securis was substantially influenced by temperature and salinity, but there was no interaction between both factors (Table 1C, Fig. 3B); their clearance rate generally decreased at low and high temperatures, and at salinities below 12‰. Temperature and salinity interactively affected the

clearance rate of B. variabilis (Table 1C, Fig. 3C) in which their clearance rate was almost totally inhibited at the low temperature treatment (14 °C) and salinities below 12‰. At the low temperature treatment (14 °C), the byssal thread production (BTP) of M. sallei was significantly reduced or inhibited (Table 1D, Fig. 3A). The number of byssal threads also differed significantly between salinities, but no clear pattern was observed (Fig. 3A). In X. securis, salinity of 7‰ significantly reduced the BTP (Table 1D, Fig. 3B). Temperature and salinity separately affected BTP in B. variabilis, which was greatly reduced at the lowest temperature (14 °C) and almost completely inhibited at salinities below 12‰ (Table 1D, Fig. 3C). Results of the permutational MANOVA test indicated that the responses (i.e., lethal and sub-lethal) were affected by the interaction of species, temperature and salinity (Table 2). The pair-wise comparison indicated that the temperature and salinity tolerances were significantly different among the three bivalve species (Fig. 4). The principal coordinates analysis indicated that both axes (PCO1 and PCO2) could jointly explain about 89% of the variation in the multivariate ordination (Fig. 4). Eigenvectors indicated that clearance rate and attachment variables could better explain the two dimensional ordination. In the ordination, there was a main group (higher number of points, Fig. 4) that represents those treatments with high responses in terms of survival, attachment, clearance rate and byssal thread production, whereas the smaller four groups represent lower responses. Xenostrobus securis is the species with more treatments within the high response group, being mainly those treatments with salinity below 17‰ in the low response groups. For M. sallei low temperature treatments (i.e., 14 °C) had the lowest responses. Brachidontes variabilis had overall more treatments with low responses, especially those with salinity treatments below 22‰ at low and high temperatures. 4. Discussion The results of this study indicated that the non-native bivalve species Xenostrobus securis and Mytilopsis sallei have higher survival and sub-lethal responses (i.e. attachment, byssal thread production and clearance rate) to environmentally relevant temperature and salinity conditions than the native Brachidontes variabilis. Xenostrobus securis and M. sallei had higher survival under all temperature and salinity treatments, whereas B. variabilis was adversely affected by high temperature and low salinities. For the sub-lethal responses, X. securis was the least affected by the temperature and salinity conditions among the

Table 1 Results of two-way ANOVAs to test the effect of temperature (14, 22 and 30 °C) and salinity (7, 12, 17, 22, 27, and 32‰) treatments on A) survival, B) attachment, C) clearance rate and D) byssal thread production of the non-native bivalves Mytilopsis sallei and Xenostrobus securis, and the native bivalve Brachidontes variabilis. On the top of each table the data transformation used for the analysis is indicated (NT = no transformation, Sqrt = square root transformation). Bold P-values indicate significant probabilities under 0.01. Mytilopsis sallei Source A) Survival Temperature Salinity Temp × Sal Error B) Attachment Temperature Salinity Temp × Sal Error C) Clearance rate Temperature Salinity Temp × Sal Error D) Byssal thread Temperature Salinity Temp × Sal Error

df 2 5 10 54 2 5 10 54 2 5 10 54 2 5 10 72

MS (NT) 0.04 0.13 0.13 0.15 (NT) 675.37 0.23 0.28 0.30 (Sqrt) 0.60 0.04 0.02 0.02 (NT) 13.52 0.76 0.27 0.18

Xenostrobus securis F

P

0.28 0.90 0.84

0.756 0.488 0.589

2244.3 0.75 0.91

b0.001 0.591 0.527

32.49 2.06 1.21

b0.001 0.085 0.306

76.18 4.30 1.53

b0.001 0.002 0.148

MS (NT) 0.68 0.49 0.18 0.13 (NT) 21.10 110.12 6.66 1.71 (NT) 0.92 0.59 0.03 0.02 (Sqrt) 1.44 3.32 0.31 0.42

Brachidontes variabilis

F

P

5.25 3.77 1.39

0.008 0.005 0.209

12.32 64.29 3.89

b0.001 b0.001 0.001

42.28 27.08 1.18

b0.001 b0.001 0.324

3.41 7.89 0.75

0.038 b0.001 0.678

MS (NT) 70.06 14.28 12.69 1.19 (NT) 33.68 244.52 8.55 1.90 (Sqrt) 1.84 0.53 0.13 0.00 (NT) 3.94 4.03 0.86 0.41

F

P

58.88 12.00 10.66

b0.001 b0.001 b0.001

17.74 128.82 4.50

b0.001 b0.001 b0.001

416.76 119.87 29.99

b0.001 b0.001 b0.001

9.68 9.90 2.12

b0.001 b0.001 0.034

Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046

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Fig. 2. Effect of 96-h acute temperature and salinity test on the survival and attachment (±95% CI) of the non-native bivalves A) Mytilopsis sallei and B) Xenostrobus securis, and the native C) Brachidontes variabilis. When ANOVA test (Table 1) indicated significant differences, a post-hoc Tukey's test was conducted and the results are shown on each graph. Numbers on the right corner indicate differences between temperature treatments. Capital letters on the bars indicate differences between salinities treatments. Lowercase letters on each bar indicate differences between temperature within each salinity group (conducted after significant interaction between temperature and salinity). The lack of numbers or letters indicates that Tukey's test was not conducted due to ANOVA results.

three species. These findings are consistent with field observations where X. securis dominates fouling communities over M. sallei and B. variabilis (Morton and Leung, 2015). The high survival and strong sub-lethal response of X. securis found in the current study could be explained by its native distribution in estuaries in Australia, where it tolerates a range of salinities from 1 to 31‰ in its natural habitat (Wilson, 1968). Water temperature in its native range fluctuates between 13 and 27 °C (Thomson et al., 2001), with few impacts on its populations (Wilson, 1969). In this study, only salinities below 12‰ reduced the attachment, byssal thread production and clearance rate of X. securis, but did not totally inhibit these responses (except at the lowest temperature and salinity). Xenostrobus securis can survive months at salinities of 1‰ (Wilson, 1969). Due to its high salinity tolerance, it has become highly invasive in estuaries in the Mediterranean and Atlantic Sea of Europe, with high abundances in hard and soft substrata where it competes and sometimes displaces the native mussel Mytilus galloprovincialis (Garci et al., 2007; Gestoso et al., 2013; Sousa et al., 2009). The high environmental tolerance and ability to colonize hard and soft substrata of X. securis suggest that it could become highly invasive in estuarine communities in Hong Kong and other coasts of the South China Sea region. In this study, M. sallei showed a high survival in all the temperature and salinity treatments but its sub-lethal responses were drastically reduced at the low temperature treatment (14 °C), indicating that its population could be indirectly affected by low clearance rate (i.e., food intake rate) or weak attachment under cold conditions. Mytilopsis sallei is a tropical species in estuaries of Central America where it can tolerate

low salinities (Puyana, 1995), which may explain the current invasive distribution in the tropical and sub-tropical Indo-Pacific region. A recruitment experiment conducted in an estuary in Xiamen (China), found that high densities of M. sallei in summer drastically drop in winter with temperatures as low as 16 °C (Cai et al., 2014). The low temperature that M. sallei could experience in Hong Kong in the winter season may limit its population growth, especially, in communities where it has to compete with X. securis. Based on the present results, B. variabilis had low survival under high temperatures (30 °C) and low salinities (b17‰), whereas its sub-lethal responses were reduced by low temperature (14 °C) and low salinities (b17‰). It could be that low temperatures induced a chill coma (Rupp and Parsons, 2004) that may result in death over a period of time longer than the 96-h of the acute test. The low tolerance and response of B. variabilis to salinities below 17‰, as shown in the current experiment, is consistent with a previous study that found low survival of B. variabilis in salinities between 0 and 16‰ (Morton and Chan, 1990). In an experiment of temperature and salinity tolerance, Ma (2013) found that the survival period of B. variabilis decreased at the combination of the high temperature treatment of 32 °C and low salinity of 8‰, whereas byssal thread production drastically decreased with 16 °C treatment and salinities below 16‰. In this study, since B. variabilis has a very low abundance in the sites where the non-native bivalve species coexist, we have to collect B. variabilis from a different site. However, our results are in good agreement with the aforementioned studies on the temperature and salinity tolerance of B. variabilis which were collected from other sites, indicating that the effect of spatial

Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046

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Fig. 3. Effect of temperature and salinity treatments on the clearance rate and byssal thread production (±95% CI) of the non-native bivalves A) Mytilopsis sallei and B) Xenostrobus securis, and the native C) Brachidontes variabilis after the 96 h acute test. Post-hoc Tukey test are indicated with numbers and letters on each graph. For details of the Tukey's test see Fig. 2.

variation may not appreciably affect the overall results. Although B. variabilis is native to the Indo-Pacific region (Bernard et al., 1993) and might be expected to be well adapted to Hong Kong's seasonal and spatial conditions, it seems that its tolerance is narrow, which may explain its low abundance in communities where X. securis and M. sallei are present (Morton and Leung, 2015). Morton and Leung (2015) found that the distribution of these three species is partitioned in the estuarine section of Shing Mun River in Hong Kong. Xenostrobus securis is distributed along the estuarine river from the upper to the lower part (through salinities on average ranging from 5 to 28‰), whereas M. sallei is only present in the upper part of the estuarine river and B. variabilis in the lower part close to the sea. The high salinity tolerance of X. securis may explain its dominance through the estuary, compared to B. variabilis that only tolerates moderately low salinities. The dominant abundance of X. securis in this estuary is only surpassed by M. sallei in the uppermost part (low salinity, ~5‰)

Table 2 Results of Permutational MANOVAs to compare the responses (i.e. survival, attachment, clearance rate and byssal thread production) of the three bivalve species under the temperature (14, 22 and 30 °C) and salinity (7, 12, 17, 22, 27, and 32‰) treatments. Source

df

MS

F

P

Species Temperature Salinity Sp × Temp Sp × Sal Temp × Sal Sp × Temp × Sal Residual

2 2 5 4 10 10 20 162

25,628 86,637 28,646 22,225 10,952 1802 1973 691

37.1 125.4 41.5 32.2 15.9 2.6 2.9

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

of the river (Morton and Leung, 2015). In the 1980's, M. sallei was a dominant fouling species in dockyards in Hong Kong under salinities of 25– 32‰ (Morton, 1989a), which suggests that X. securis is likely displacing M. sallei to areas with the lower salinities. Due to the strong seasonal variation experienced in Hong Kong, further tolerance experiments may include seasonality to better understand the role of temporal variation on the non-native bivalve tolerance and their distribution in Hong Kong. A high availability of resources (e.g. substrata and food) is considered crucial to allow species to become invasive (Shea and Chesson, 2002). Non-native species likely become dominant in estuaries and bays under environmental disturbances, where non-native species may find a niche with lower competition and predation pressure (Astudillo et al., 2016; Cheng and Hovel, 2010; Osman et al., 2010). Bivalves as filter-feeders find high amounts of food in estuaries and human-disturbed environments due to eutrophication and heavy loads of organic matter (Morton, 1989b). The high clearance rates (i.e., high food intake rate) of X. securis under different temperature and salinity conditions could explain its increasing population in Hong Kong during the last few years. A release of predation due to unfavorable environmental conditions (Cheng and Hovel, 2010) or low preference by native predators (Veiga et al., 2011) in estuaries could also explain the restricted distribution of non-native species to estuarine environments in Hong Kong. Hence, predation should also be taken into account in future studies to understand the abundance and potential distribution of these species in the South China Sea. 5. Conclusion In summary, this study demonstrates that the recently introduced bivalve Xenostrobus securis has a wider temperature and salinity

Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046

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Fig. 4. Principal coordinates analysis (PCO) for the responses of the non-native bivalves Mytilopsis sallei and Xenostrobus securis, and the native Brachidontes variabilis under different temperature and salinity treatments. Pair-wise comparison for differences between species is indicated on the right of the figure. The average of survival (S), attachment (A), clearance rate (C), and byssal thread production (B) was used for each combination of temperature and salinity treatment. Species are differentiated by color, temperature treatments by shape of the plot, and salinity by the number close to each plot. Eigenvectors indicate the contribution of each variable to explain the ordination. The total variation explained by the PCO is indicated in each axis.

tolerance than the previously introduced non-native Mytilopsis sallei (introduced ~ 30 years ago) and the native Brachidontes variabilis under Hong Kong seawater conditions. Since X. securis is a bioengineer species that can modify hard and soft bottom habitats (Barbieri et al., 2011), they could become a threat for biodiversity in Hong Kong. Xenostrobus securis is currently found on fouling communities in Hong Kong, however, a monitoring in estuarine rocky shores and sediments (e.g. mangroves) must be conducted to determine their impact in natural benthic habitats. Xenostrobus securis and M. sallei have become invasive in other regions impacting clam and mussel farming industries (Garci et al., 2007; Liao et al., 2010). In the Pearl River estuary in Hong Kong, oyster culture is one of the main activities for fishermen. The potential of non-native species to compete for resources with oysters, or overgrow their shells (especially for juvenile oysters) should be considered in an evaluation of their economic impact. Acknowledgments The authors sincerely thank the Environment and Conservation Fund (ECF Project: 2010-28) for partially supporting this study. The authors would also like to thank the staff of the Swire Institute of Marine Science for their assistance in this project and to Angie Chan for her help with the laboratory work. We are grateful to Dr. Nicolas Ory for providing comments on an early draft of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2017.02.046. References Abdel-Razek, F.A., Chiba, K., Kurokura, H., Okamoto, K., Hirano, R., 1993. Life history of Limnoperna fortunei kikuchii in Shonai Inlet, Lake Hamana. Suisanzoshoku 41, 97–104. Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods. PRIMER-E Plymouth, UK. Astudillo, J.C., Wong, J.C.Y., Dumont, C., Bonebrake, T.C., Leung, K.M.Y., 2014. Status of six non-native marine species in the coastal environment of Hong Kong, 30 years after their first record. Bioinvasions Rec. 3, 123–137. Astudillo, J.C., Leung, K.M.Y., Bonebrake, T.C., 2016. Seasonal heterogeneity provides a niche opportunity for ascidian invasion in subtropical marine communities. Mar. Environ. Res. http://dx.doi.org/10.1016/j.marenvres.2016.09.001.

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Please cite this article as: Astudillo, J.C., et al., The recently introduced bivalve Xenostrobus securis has higher thermal and salinity tolerance than the native Brachidontes variabi..., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.046